Affinage

SIX5

Homeobox protein SIX5 · UniProt Q8N196

Length
739 aa
Mass
74.6 kDa
Annotated
2026-06-10
34 papers in source corpus 18 papers cited in narrative 17 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

SIX5 (DMAHP) is a homeodomain transcription factor that acts as a sequence-specific transcriptional activator and is the gene most proximal to the DM1 (myotonic dystrophy type 1) CTG repeat locus, where expansion eliminates a DNase I-hypersensitive enhancer and reduces SIX5 transcription in cis, most severely from the expanded allele (PMID:9241282, PMID:9241283, PMID:9817928). SIX5 binds DNA through its homeodomain, recognizing the Na+/K+-ATPase alpha-1 (ATP1A1) regulatory element, with the SIX-domain-plus-homeodomain protein forming a second, dimer-like complex at the same site (PMID:10756185). It activates transcription of target genes including Igfbp5 (PMID:11978764), and assembles into transcriptional activator complexes with EYA1 — an interaction disrupted by SIX5 missense mutations identified in branchio-oto-renal (BOR) syndrome patients (PMID:17357085). Loss-of-function mouse studies establish SIX5 as a dosage-sensitive contributor to specific DM1 features: heterozygous and homozygous Six5 disruption causes cataracts associated with elevated Na+/K+-ATPase alpha-1 protein and altered lens ion homeostasis (PMID:10802667, PMID:10802668), infraHisian cardiac conduction delay (PMID:12397222), and male sterility with impaired spermatogenesis, Leydig cell hyperproliferation, and reduced testicular c-Kit (PMID:15163633). Genetic dissection places SIX5 outside the DM1 features driven by Na+ channel gating, cognition, and motivation, which track instead with DMPK and MBNL1 (PMID:11526199, PMID:20360842). In dystrophic muscle, combined reduction of Six4/Six5 dosage enhances regeneration (PMID:27224259), and in cancer SIX5 functions in EYA3-p300 activator complexes and as a direct transcriptional activator of growth- and invasion-associated genes (PMID:35957720).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 1997 High

    Established why SIX5 is implicated in DM1: the CTG expansion does not merely affect DMPK but eliminates an upstream enhancer that drives SIX5/DMAHP transcription in cis.

    Evidence DNase I hypersensitivity mapping and allele-specific RT-PCR in DM patient cells across two independent labs

    PMID:9241282 PMID:9241283

    Open questions at the time
    • Did not establish which downstream phenotypes are attributable to SIX5 loss versus other DM1 mechanisms
    • Mechanism of chromatin compaction over the enhancer not resolved
  2. 1998 Medium

    Defined the SIX5 promoter architecture, identifying Sp1/Sp3 positive elements and confirming that transcription start sites lie downstream of the CTG repeat, excluding CUG repeats from the mRNA.

    Evidence Transcription start site mapping and promoter-reporter deletion analysis in P19 cells

    PMID:9817928

    Open questions at the time
    • Tissue-specific regulation only partially mapped
    • Identity of the novel negative regulatory factor unknown
  3. 2000 Medium

    Identified a direct DNA target and binding mode for SIX5, showing it engages the ATP1A1 regulatory element through its homeodomain, linking the factor to ion-transport gene regulation.

    Evidence GST-fusion recombinant protein and EMSA with defined oligonucleotides

    PMID:10756185

    Open questions at the time
    • In vitro binding not confirmed by in vivo occupancy
    • No binding detected at DMPK promoter sites — broader target repertoire unresolved
    • Single lab
  4. 2000 High

    Demonstrated causally that SIX5 loss produces a discrete DM1 phenotype, with dosage-dependent cataracts linked to elevated Na+/K+-ATPase alpha-1 and altered lens ion homeostasis.

    Evidence Six5 beta-gal knock-in mice, dosage series, histology, Western blot, RT-PCR across two labs

    PMID:10802667 PMID:10802668

    Open questions at the time
    • No skeletal muscle phenotype detected, narrowing SIX5's role within DM1
    • Direct mechanistic link between SIX5 and ATP1A1 regulation in lens not established in vivo
  5. 2001 Medium

    Excluded SIX5 from the Na+ channel gating abnormality of DM1 myotonia, refining which features are SIX5-dependent.

    Evidence Patch-clamp recordings from Six5+/- versus Dmpk+/- skeletal muscle

    PMID:11526199

    Open questions at the time
    • Negative result; does not address other muscle phenotypes
  6. 2002 Medium

    Established SIX5 as a transcriptional activator and identified Igfbp5 as a direct target confirmed by both gain- and loss-of-function.

    Evidence VP16-Six5 overexpression with microarray, RT-PCR validation, and Six5-/- fibroblast analysis

    PMID:11978764

    Open questions at the time
    • Most of the 21 candidate targets not validated as direct
    • Co-activators driving native activation not defined
  7. 2002 Medium

    Linked SIX5 dosage to the infraHisian cardiac conduction defects seen in DM1.

    Evidence In vivo electrophysiology and echocardiography in Six5+/- mice

    PMID:12397222

    Open questions at the time
    • Transcriptional targets responsible for the conduction phenotype unknown
    • Single lab
  8. 2004 Medium

    Defined a SIX5-dependent male fertility program, implicating c-Kit downregulation in spermatogenic cell apoptosis.

    Evidence Six5-/- mouse phenotyping, TUNEL, Western blot, hormone assays

    PMID:15163633

    Open questions at the time
    • c-Kit not shown to be a direct SIX5 target
    • Cause of Leydig cell hyperproliferation unresolved
  9. 2007 Medium

    Connected SIX5 to a partner-dependent activator complex and to a second human disease, showing BOR syndrome mutations impair EYA1-SIX5 binding and transactivation.

    Evidence Patient mutation screening with interaction and transcription activation assays

    PMID:17357085

    Open questions at the time
    • Target genes of the EYA1-SIX5 complex not identified
    • Single study
  10. 2010 Medium

    Placed SIX5 outside the cognitive/motivational axis of DM1, which tracks with MBNL1 instead.

    Evidence Parallel behavioral testing of Six5-/-, Dmpk-/-, and Mbnl1-/- mice

    PMID:20360842

    Open questions at the time
    • Negative result for behavior; does not exclude subtler neural roles
  11. 2016 Medium

    Showed SIX4/SIX5 normally limit regenerative capacity in dystrophic muscle, since reduced dosage improves mdx phenotype and lifespan.

    Evidence Six4/Six5 double-heterozygous mdx mice with histomorphometry and regeneration markers

    PMID:27224259

    Open questions at the time
    • SIX4 and SIX5 reduced together, so individual contribution unresolved
    • Direct regenerative target genes not identified
  12. 2022 Medium

    Extended SIX5 function to oncogenic transcription, defining an EYA3-SIX5-p300 complex that transactivates EGFR, VEGFD, and MMP genes to drive colorectal tumor growth.

    Evidence Co-IP, mass spectrometry, ChIP, knockdown/overexpression, xenograft

    PMID:35957720

    Open questions at the time
    • Whether the same complex operates in normal tissues unknown
    • Single lab
  13. 2025 Low

    Implicated SIX5 as a direct transcriptional activator of pro-tumorigenic targets (EXO1, UBE2C) downstream of KDM5C in glioblastoma.

    Evidence ChIP, dual-luciferase reporter, knockdown/overexpression, rescue, xenograft

    PMID:40946999 PMID:41939887

    Open questions at the time
    • Single lab, no replication
    • Mechanism of KDM5C-SIX5 regulation not detailed
    • Direct relevance to canonical SIX5 developmental function unclear

Open questions

Synthesis pass · forward-looking unresolved questions
  • The genome-wide native SIX5 target repertoire and the co-activator requirements that determine context-specific (lens, testis, heart, cancer) gene programs remain undefined.
  • No unbiased in vivo ChIP-seq target map
  • How EYA partner choice (EYA1 vs EYA3) directs distinct programs is unknown
  • Structural basis of SIX-domain dimerization on DNA not solved

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140110 transcription regulator activity 3 GO:0003677 DNA binding 1
Localization
GO:0005654 nucleoplasm 1
Pathway
R-HSA-74160 Gene expression (Transcription) 3
Partners
EP300EYA1EYA3
Complex memberships
EYA1-SIX5 transcriptional activator complexEYA3-SIX5-p300 complex

Evidence

Reading pass · 17 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1997 CTG trinucleotide repeat expansion at the DM1 locus eliminates a DNase I-hypersensitive site containing an enhancer element that regulates DMAHP/SIX5 transcription, reducing steady-state DMAHP transcript levels 2- to 4-fold overall and greatly reducing transcripts from the expanded allele specifically. DNase I hypersensitivity assay, allele-specific RT-PCR in DM patient cells Nature genetics High 9241282 9241283
2000 Loss-of-function of Six5 in mice (homozygous knockout replacing exon 1 with beta-galactosidase reporter) causes lenticular opacities (cataracts) at a higher rate than controls, with reporter expression confirmed in the developing lens; no skeletal muscle abnormalities were detected. Targeted gene disruption (beta-gal knock-in), histological and functional phenotyping of Six5-/- mice Nature genetics High 10802667 10802668
2000 Cataract formation in Six5-deficient mice is inversely related to Six5 dosage (heterozygous Six5+/- mice also develop cataracts), and Six5+/- and Six5-/- mice show increased steady-state Na+/K+-ATPase alpha-1 subunit protein levels and decreased Dm15 mRNA levels, implicating altered ion homeostasis in lens pathology. Dosage analysis of Six5+/- and Six5-/- mice, Western blotting, RT-PCR Nature genetics High 10802668
2000 SIX5 protein binds the Na+/K+-ATPase alpha-1 subunit gene (ATP1A1) regulatory element (ARE) — the same site as murine Six4 — through its homeodomain; the protein containing both the SIX domain and homeodomain forms a second (presumed dimer) complex with the ARE. No binding was detected to two putative sites in the DMPK promoter. GST fusion protein expression, gel retardation (EMSA) assays with double-stranded oligonucleotides Nucleic acids research Medium 10756185
2000 SIX5 protein localizes to the nucleoplasm with a granular distribution in HeLa cells; endogenous SIX5 migrates at ~100 kDa in SDS-PAGE. A shorter splice isoform mRNA exists but the corresponding shorter protein was undetectable, suggesting the full-length isoform is the major functional protein. Monoclonal antibody panel (18 mAbs), Western blotting, immunolocalization in HeLa cells, phage-display epitope mapping Journal of cellular biochemistry Medium 15962300
2002 Six5 functions as a transcriptional activator; overexpression of constitutively active VP16-Six5 in P19 cells identified 21 upregulated target genes including Igfbp5. Igfbp5 was confirmed as a direct Six5 transcriptional target, and its overall expression was decreased in Six5-deficient mouse fibroblasts. Adenovirus-mediated VP16-Six5 overexpression, cDNA microarray profiling, validation by RT-PCR, analysis in Six5-/- fibroblasts Human molecular genetics Medium 11978764
2004 Six5 knockout mice show male sterility with progressive loss of testicular mass, defects in spermatogenic cell survival and spermiogenesis, Leydig cell hyperproliferation, elevated intra-testicular testosterone, and reduced steady-state c-Kit levels in the testis, suggesting c-Kit downregulation contributes to elevated spermatogenic cell apoptosis. Six5-/- mouse phenotyping, histology, TUNEL assay, Western blotting, hormone assays Human molecular genetics Medium 15163633
2002 Six5 heterozygous mice exhibit prolonged QRS duration and delayed infraHisian conduction compared to wild-type, as well as enlarged left ventricular end-diastolic dimension, suggesting Six5 loss contributes to infraHisian conduction delay. In vivo electrophysiologic studies, echocardiography, heart rate variability testing in Six5+/- mice Journal of interventional cardiac electrophysiology Medium 12397222
2001 Six5+/- mice do not exhibit late Na+ channel burst activity (a hallmark of myotonic dystrophy seen in Dmpk+/- mice), and Na+ current amplitude is unchanged, demonstrating that Six5 deficiency does not contribute to the Na+ channel gating abnormality. Cell-attached patch clamp recordings from skeletal muscle of Six5+/- mice vs. wild-type and Dmpk+/- mice Physiological genomics Medium 11526199
2007 Missense mutations in SIX5 identified in BOR syndrome patients impair EYA1-SIX5 protein binding and reduce the ability of SIX5 or the EYA1-SIX5 complex to activate gene transcription, establishing that SIX5 functions with EYA1 as a transcriptional activator complex. Patient mutation screening, functional transcription activation assays, protein-protein interaction assays for EYA1-SIX5 binding American journal of human genetics Medium 17357085
1998 The mDMAHP/Six5 promoter contains multiple transcription initiation sites (one proximal site specific to early embryo E11, two shared among heart, skeletal muscle, and embryo); positive regulatory elements are Sp1/Sp3 binding sites and a novel factor binding site acts as a negative element. All initiation sites lie downstream of the CTG repeat locus, excluding CUG repeats from the mRNA. Primer extension/S1 nuclease mapping of transcription start sites, promoter-reporter deletion analysis in P19 cells, Sp1/Sp3 binding site identification Human molecular genetics Medium 9817928
2010 Behavioral and cognitive testing of Six5-/- mice showed no motivational deficits or spatial learning/memory abnormalities, while Mbnl1-/- mice did exhibit these deficits, placing SIX5 outside the pathway responsible for cognitive and motivational features of DM1. Open field, elevated plus maze, Morris water maze, fear conditioning, sucrose consumption assays in Six5-/-, Dmpk-/-, and Mbnl1-/- mice PloS one Medium 20360842
2016 Reduced Six4 and Six5 gene dosage (Six4+/-5+/-) in mdx mice improves dystrophic phenotype (fewer small myofibers, lower serum creatine kinase/lactate dehydrogenase) and prolongs lifespan by 33.8%, associated with enhanced muscle regeneration markers (MYOD1, MYOG, SIX1 positive cells), indicating SIX4/SIX5 normally limit regenerative capacity in dystrophic muscle. Double heterozygous Six4/Six5 knockout in mdx background, histomorphometry, serum enzyme assays, immunohistochemistry for regeneration markers, grip strength and treadmill testing Development, growth & differentiation Medium 27224259
2022 SIX5 forms a complex with hypoxia-induced EYA3 and histone acetyltransferase p300 in colorectal cancer cells; this EYA3-SIX5-p300 complex binds the promoters of EGFR, VEGFD, and five MMPs (MMP3, MMP7, MMP8, MMP21, MMP26) to transactivate them, promoting tumor cell growth. Co-immunoprecipitation, mass spectrometry, ChIP assay, knockdown/overexpression functional assays, xenograft model Annals of translational medicine Medium 35957720
2022 SIX5 transcriptionally activates LINC01468 by binding its promoter in lung adenocarcinoma cells, and SIX5 overexpression exacerbates LUAD cell proliferation, migration, and invasion. ChIP, luciferase reporter assay, RT-qPCR, knockdown/overexpression, xenograft model Cell death & disease Low 35387981
2025 SIX5 directly binds the EXO1 promoter and transcriptionally activates EXO1 expression in glioblastoma cells, promoting tumor cell migration and angiogenesis; KDM5C positively regulates SIX5 expression upstream. ChIP, dual-luciferase reporter assay, SIX5 knockdown/overexpression, xenograft model Brain research Low 40946999
2026 KDM5C positively regulates SIX5 expression; SIX5 directly binds the UBE2C promoter to activate its transcription, thereby activating AKT/mTOR signaling and upregulating glycolytic enzymes (GLUT1, HK2, PGK1, LDHA) in glioblastoma. ChIP, luciferase reporter, lentiviral knockdown/overexpression, rescue experiments, xenograft model Frontiers in immunology Low 41939887

Source papers

Stage 0 corpus · 34 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1997 Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP. Nature genetics 194 9241282
2000 Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nature genetics 179 10802667
1997 Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. Nature genetics 178 9241283
2007 Transcription factor SIX5 is mutated in patients with branchio-oto-renal syndrome. American journal of human genetics 138 17357085
2000 Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nature genetics 136 10802668
2015 The AVR2-SIX5 gene pair is required to activate I-2-mediated immunity in tomato. The New phytologist 86 25967461
2011 Mutation screening of the EYA1, SIX1, and SIX5 genes in a large cohort of patients harboring branchio-oto-renal syndrome calls into question the pathogenic role of SIX5 mutations. Human mutation 80 21280147
2018 The Fusarium oxysporum Avr2-Six5 Effector Pair Alters Plasmodesmatal Exclusion Selectivity to Facilitate Cell-to-Cell Movement of Avr2. Molecular plant 78 29481865
1999 Characterization of the expression of DMPK and SIX5 in the human eye and implications for pathogenesis in myotonic dystrophy. Human molecular genetics 63 9949207
2001 Drosophila homolog of the myotonic dystrophy-associated gene, SIX5, is required for muscle and gonad development. Current biology : CB 45 11470409
2004 Six5 is required for spermatogenic cell survival and spermiogenesis. Human molecular genetics 40 15163633
2013 Mutational analysis of EYA1, SIX1 and SIX5 genes and strategies for management of hearing loss in patients with BOR/BO syndrome. PloS one 36 23840632
2002 Characterization of cardiac conduction system abnormalities in mice with targeted disruption of Six5 gene. Journal of interventional cardiac electrophysiology : an international journal of arrhythmias and pacing 33 12397222
2001 Effect of triplet repeat expansion on chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy. Molecular genetics and metabolism 33 11592825
1999 Myotonic dystrophy: tissue-specific effect of somatic CTG expansions on allele-specific DMAHP/SIX5 expression. Human molecular genetics 30 10332033
2012 Mutation screening of the EYA1, SIX1, and SIX5 genes in an East Asian cohort with branchio-oto-renal syndrome. The Laryngoscope 28 22447252
2010 Muscleblind1, but not Dmpk or Six5, contributes to a complex phenotype of muscular and motivational deficits in mouse models of myotonic dystrophy. PloS one 27 20360842
2002 Identification of transcriptional targets for Six5: implication for the pathogenesis of myotonic dystrophy type 1. Human molecular genetics 24 11978764
2000 Functional analysis of the homeodomain protein SIX5. Nucleic acids research 24 10756185
2022 SIX5-activated LINC01468 promotes lung adenocarcinoma progression by recruiting SERBP1 to regulate SERPINE1 mRNA stability and recruiting USP5 to facilitate PAI1 protein deubiquitylation. Cell death & disease 23 35387981
2004 UNC-39, the C. elegans homolog of the human myotonic dystrophy-associated homeodomain protein Six5, regulates cell motility and differentiation. Developmental biology 20 15282156
2000 Expression of a homeobox gene (SIX5) in borderline ovarian tumours. Journal of clinical pathology 17 10823141
2022 The primary function of Six5 of Fusarium oxysporum is to facilitate Avr2 activity by together manipulating the size exclusion limit of plasmodesmata. Frontiers in plant science 16 35968143
2000 Reduced expression of DMAHP/SIX5 gene in myotonic dystrophy muscle. Muscle & nerve 16 10951446
1998 Promoter of mDMAHP/Six5: differential utilization of multiple transcription initiation sites and positive/negative regulatory elements. Human molecular genetics 13 9817928
2022 Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression. Annals of translational medicine 11 35957720
1999 Reduction of the DM-associated homeo domain protein (DMAHP) mRNA in different brain areas of myotonic dystrophy patients. Neuromuscular disorders : NMD 11 10399747
2016 Low Six4 and Six5 gene dosage improves dystrophic phenotype and prolongs life span of mdx mice. Development, growth & differentiation 6 27224259
2001 Skeletal muscle Na currents in mice heterozygous for Six5 deficiency. Physiological genomics 4 11526199
2023 The SIX5 Protein in Fusarium oxysporum f. sp. cepae Acts as an Avirulence Effector toward Shallot (Allium cepa L. Aggregatum Group). Microorganisms 3 38138005
2022 Identification of homozygous missense variant in SIX5 gene underlying recessive nonsyndromic hearing impairment. PloS one 3 35709191
2005 Characterisation of the transcription factor, SIX5, using a new panel of monoclonal antibodies. Journal of cellular biochemistry 3 15962300
2026 KDM5C-regulated SIX5 promotes glioblastoma progression through transcriptional activation of UBE2C and enhancement of the Warburg effect. Frontiers in immunology 0 41939887
2025 Role of SIX5-mediated EXO1 overexpression in driving glioblastoma progression: Insights into tumor cell migration and angiogenesis. Brain research 0 40946999

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